Vitamin K-dependent proteins are a class of proteins involved in maintaining hemostasis. The dependency on vitamin K occurs during the biosynthesis of the proteins, as vitamin K is a cofactor in the carboxylation of glutamic acid residues of the proteins. The result of this reaction is the formation of a γ-carboxyglutamate (gamma-carboxyglutamate), referred to as gla residue. The formation of gla residues within several proteins of the blood clotting cascade is critical for their normal function. The presence of gla residues allows the protein to chelate divalent cations, e.g. calcium, and thereby render an altered conformation, an alteration of the surface charges on the protein, and thus, an alteration of the biological activity of the protein.
EP0363126 utilizes said changes in the protein in the presence of divalent cations to selectively purify recombinant vitamin K-dependent proteins. The method is based on conventional ion exchange chromatography to separate the vitamin K-dependent proteins based on the ionically altered binding affinity to the substrate in the absence or presence of divalent cations and is called “pseudo-affinity chromatography”. Thus, the method described in EP0363126 allows to select recombinant vitamin K-dependent proteins based on their quantity of gla residues.
U.S. Pat. No. 5,714,583 describes purification methods for rFIX using anion exchange chromatography in the pseudo-affinity mode to increase the purity of rFIX. Selected inactive forms of FIX and contaminating host cell proteins remain bound to the column, while active FIX and some less active forms are eluted by the addition of a divalent cation to the buffer.
EP0363126 as well as U.S. Pat. No. 5,714,583 are aimed on the purification of recombinant protein molecules with an increased biological activity.
However, the therapeutic efficacy depends not only on the biological activity of a recombinant protein, but also on the in vivo recovery.
This can be seen from the following example: Hemophilia B, a hereditary recessive bleeding disorder, is successfully treated by replacement therapy consisting of the administration of preparations of human plasma derived (pdFIX) or recombinant blood coagulation factor IX (rFIX). The commercially available recombinant product, which is marketed under the trade name Benefix™, is manufactured by using stable transfected Chinese hamster ovary (CHO) cells co-expressing rFIX together with endopeptidase PACE/Furin, and is highly purified via multiple filtration and chromatographic steps (Kaufman et al., 1986; Wasley et al., 1993; Harrison et al., 1998). In clinical studies, Benefix™ has been shown to be safe and effective, but a 20 to 50% higher dosage than for pdFIX is needed for successful treatment. This is due to a 30 to 50% lower in vivo recovery for CHO derived rFIX than for pdFIX, as revealed by pharmacokinetic data collected from pre-clinical and clinical studies, where pdFIX and rFIX are compared in different animal models (Keith, Jr. et al., 1995; Brinkhous et al., 1996; Schaub et al., 1998; McCarthy et al., 2002), and clinical studies in hemophilia B patients (Keith, Jr. et al., 1995; White et al., 1997; White et al., 1998; Bjorkman et al., 2001; Roth et al., 2001; Ewenstein et al., 2002; Poon et al., 2002; Ragni et al., 2002; Kisker et al., 2003; Shapiro et al., 2005a). The circulating half-life of rFIX is not distinguishable from pdFIX preparations. Biochemical comparison between pdFIX and CHO derived rFIX revealed differences in post-translational modifications (Bond et al., 1998). The lower degree of phosphorylation of a unique site at the activation-peptide amino acid serine 158 and the lower degree of sulfation of tyrosine 155 have been assigned to the lower in-vivo recovery of rFIX (White et al., 1997; Kaufman, 1998), although experimental evidence to proof this assumption has not been published to-date. These two modifications were identified to occur at less than 15% for the tyrosine-sulfation and at less than 1% for the serine phosphorylation in the recombinant protein, whereas the plasma derived protein has both modifications to more than 90% completed. Similar pharmacokinetic properties to Benefix™ were found for myotube-synthesized rFIX after adeno-associated viral vector mediated gene delivery in a mouse model (Arruda et al., 2001).
Post-translational modifications such as sulfation and/or phosphorylation are affected by the cell line as well as the culture conditions used in the large scale production of recombinant proteins (Kaufman, 1998).
Therefore, a strong need exists for purification methods of rFIX from cell culture supernatants which allow an enrichment of molecules which have been post-translationally modified by sulfation and/or phosphorylation. Such new purification methods would allow to provide rFIX preparations which have an improved in vivo recovery and thus, could be administered in lower doses than the preparations used in the prior art.